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. 1997 Apr 15;17(8):2746-55.
doi: 10.1523/JNEUROSCI.17-08-02746.1997.

Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage

S Nogawa  1 , F Zhang, M E Ross, C Iadecola
Affiliations

Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage

S Nogawa et al. J Neurosci. .

Abstract

Cyclo-oxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, is induced during inflammation and participates in inflammation-mediated cytotoxicity. Cerebral ischemia is followed by an inflammatory reaction that plays a role in the evolution of the tissue damage. We studied whether COX-2 is induced after cerebral ischemia and if so, whether such expression contributes to ischemic brain damage. The middle cerebral artery was occluded in rats, and the ischemic area was sampled for analysis 3-96 hr later. COX-2 mRNA was determined by the competitive reverse-transcription PCR. COX-2 mRNA was upregulated in the ischemic hemisphere, but not contralaterally, beginning 6 hr after ischemia. The upregulation reached a maximum at 12 hr, at which time a fivefold induction of the message occurred. Twenty-four hours after ischemia, the concentration of prostaglandin E2 was elevated in the injured brain by 292 +/- 57% (n = 6). COX-2 immunoreactivity was observed in neurons at the medial edge of the ischemic area. Administration of the COX-2 inhibitor NS-398 attenuated the elevation in prostaglandin E2 in the postischemic brain and reduced the volume of the infarct by 29 +/- 6% (p < 0.05). Thus, cerebral ischemia leads to upregulation of COX-2 message, protein, and reaction products in the injured hemisphere. The data implicate COX-2 in the mechanisms of delayed neuronal death at the infarct border and provide the rationale for neuroprotective strategies employing COX-2 inhibitors.

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Figures

Fig. 2.
Fig. 2.
Competitive PCR to quantify the magnitude of COX-2 expression after cerebral ischemia. Samples were obtained from animals killed 12 hr after stroke. A, Competition between the COX-2 PCR product (S) and increasing amounts of a construct (C) produced by deletion of an internal portion of the COX-2 PCR product. Notice that a higher amount of construct is needed to compete out the COX-2 PCR product on the stroke side than on the contralateral side. B, Quantitative analysis of the gel presented in A. The log of the ratio of the density (COX-2/construct) was plotted as a function of the log of the concentration of the construct and fitted by linear regression analysis. The 0 value of the log of the ratio (COX-2/construct) (y-axis) represents the point at which the COX-2 PCR product and construct are present in equal amounts. Therefore, the amount of the construct corresponding to the 0 ratio (x-axis) corresponds to the amount of the COX-2 PCR product before PCR amplification. C, Group data on COX-2 expression in sham-operated rats and in rats 12 hr after transient MCA occlusion. In sham-operated rats (n = 4), COX-2 does not differ between sides. After ischemia (n = 4), there is a marked increase in the COX-2 PCR product.
Fig. 1.
Fig. 1.
A, Effect of transient focal ischemia on COX-2 mRNA expression detected by RT-PCR. PCR products were run on a gel, and the optical density of the bands was measured by image analysis. The density of the COX-2 band was divided by the density of the band of a ubiquitous gene, PBD, used as a normalization factor. Each time point represents the average of four rats. The COX-2 signal increases at 6 hr, reaches a peak at 12–24 hr, and returns to baseline at 48–96 hr. No changes in COX-2 expression are seen in the brain contralateral to the stroke. B, In contrast to COX-2, COX-1 mRNA does not increase after cerebral ischemia on either side of the brain. The density of the COX-1 band was normalized by the PBD band as described in A. The fact that the mRNA for COX-1, an enzyme closely related to COX-2, was not increased attests to the selectivity of the COX-2 upregulation and to the selectivity of the RT-PCR technique used in the present study. C, Comparison of the time course of COX-2 and iNOS expression after transient cerebral ischemia. Data are presented as δ, obtained by subtracting the density of the COX-2 or iNOS bands in the contralateral nonischemic side from that of the stroke side. The time course of COX-2 and iNOS expression in the postischemic period is similar.
Fig. 3.
Fig. 3.
Effect of focal cerebral ischemia on COX-2 immunoreactivity in paraffin-embedded sections (7 μm thickness) of rats subjected to a 2 hr occlusion of the MCA. A, Some COX-2-immunoreactive cells are located in the intact cingulate cortex medial to ischemic lesion. This region corresponds to the border zone between the anterior cerebral artery and MCA. The arrowspoint to the midline (intrahemispheric fissure); thearrowheads point to unstained neurons in the contralateral side. B, High-power view of the cells depicted in A. These cells have the morphological characteristics of normal neurons, with a large round nucleus and a prominent nucleolus. The immunoreactivity is characteristically perinuclear (cf. Yamagata et al., 1993). C, Other COX-2-positive cells are located in the transitional region between normal and infarcted brain. These cells have an angular appearance, with a shrunken cytoplasm and nucleus. In alternate sections stained with hematoxylin and eosin, these cells correspond to neurons exhibiting distinct ischemic changes (“red neurons”). Therefore, COX-2 is expressed also in injured neurons at the periphery of the ischemic territory. The region in which these COX-2 neurons are located corresponds to the so-called ischemic penumbra. The positive cells observed within the infarct are most likely shrunken neurons.P, Anatomical location of the ischemic penumbra;i, infarct. D, High-power view of the COX-2-immunoreactive neurons depicted in C. Notice the cell shrinkage. Scale bars: A, C, 500 μm; B, D, 150 μm.
Fig. 4.
Fig. 4.
Spatial distribution of COX-2-immunoreactive cells throughout the brain after transient occlusion of the MCA. The diagram depicts data from four rats. The black area represents the region of infarction identified by counterstaining the sections processed for COX-2 immunocytochemistry with hematoxylin and eosin. The darker and lighter shades of gray indicate, respectively, higher and lower density and stain intensity of positive cells. The majority of COX-2-immunoreactive neurons are located medial to the infarct in a region corresponding to the border zone of the territories of the anterior cerebral artery and MCA. Some neurons are located in the normal cortex medial to the infarcted region (cingulate cortex). Other neurons are located at the transition between normal and infarcted tissue (see Fig. 3). An increased number of COX-2-immunoreactive cells is also observed in the ipsilateral piriform cortex. In this region, the cells closer to the rhinal fissure are near the inferolateral aspect of the cortical infarct. Rare lightly stained neurons are seen in the medial border of the striatal infarct. Therefore, the majority of COX-2 positive neurons are located at the medial border of the cortical infarct.
Fig. 5.
Fig. 5.
A, Effect of transient MCA occlusion on (PGE2) in the postischemic brain. In sham-operated rats, low levels of PGE2 are present in the brain. Cerebral ischemia increases PGE2 concentration 24 hr after stroke only on the ischemic side (p < 0.05, t test; n = 6).B, Effect of the COX-2 inhibitor NS-398 on postischemic increase in PGE2 in the injured brain. NS-398 (20 mg/kg, i.p.) was administered starting 6 hr after transient MCA occlusion. Rats were killed 24 hr after ischemia. At the time of death, the rats had received three doses. NS-398 attenuates the postischemic increases in PGE2 (p < 0.05 from vehicle; ANOVA and Tukey’s test). The residual increase in PGE2after NS-398 did not reach statistical significance (p > 0.05). NS-398 slightly reduced resting levels of PGE2 in the cerebral cortex contralateral to the stroke. However, such reduction did not reach statistical significance (p > 0.05). C, Effect of NS-398 on the volume of the infarct produced by transient MCA occlusion in the rat. Rats were treated for 3 d (20 mg/kg, i.p., twice per day) starting 6 hr after induction of ischemia. NS-398 reduced the volume of the infarct in the cerebral cortex but not in the striatum. The reduction in infarct volume persists after correction for ischemic swelling [Cortex (E.C.)], suggesting that the reduction in the lesion volume is not attributable to an effect of NS-398 on ischemic edema.
Fig. 6.
Fig. 6.
Arterial pressure, plasma glucose, and rectal temperature of the rats in which the effect of NS-398 on cerebral ischemic damage was studied (see Fig. 5C). NS-398 does not affect these parameters at any of the time points studied (p > 0.05; t test from vehicle).
Fig. 7.
Fig. 7.
Spatial distribution of the infarct produced by transient MCA occlusion in vehicle-treated rats and in rats treated with the COX-2 inhibitor NS-398. In NS-398-treated rats, the lesion is smaller at all rostro-caudal levels. The area “rescued” from infarction involves primarily the medial edge of the lesion and includes the region in which the COX-2-positive neurons are located (cf. Fig. 4)
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